Mechanical loading maintains healthy cartilage while excess loading can lead to joint pathology. Crystal formation within the cartilage can also lead to osteoarthritis. Mechanical stimulation has been shown to cause a rise in intracellular Ca2+. We now show that mechanical stimulation also leads to release of ATP, an important extracellular signaling molecule. In cartilage, extracellular ATP is cleaved to AMP and PPi. PPi is involved in calcium pyrophosphate dihydrate (CPPD) crystal deposition disorders and contributes to the progression of osteoarthritis. As a signaling molecule, ATP binds to purinergic (P2Y2) receptors on chondrocytes causing a G protein-mediated rise in intracellular Ca2+. Our central hypothesis is that extracellular signaling molecules, such as ATP, are released in response to mechanical stimuli and play a major role in the metabolic response of chondrocytes to mechanical load. Our studies will use three dimensional pellet cultures formed from enzymatically isolated chondrons (chondrocytes with their native pericellular matrix). The main advantage of the chondron pellet culture system is substantial matrix deposition and assembly while the differentiated chondrocyte phenotype is maintained. The instrumentation used to apply compression to samples is an accessory of the Flexercell Strain Unit. The Compression Plus instrument uses positive pressure to produce cyclic compressive loading of samples. Initial studies will be done with porcine articular cartilage and then repeated with human articular cartilage. Specific Aim 1 is to determine the dose response of ATP release induced by cyclic compressive loading. There is now indication in the literature that ATP release is through specific channels but cell lysis must also be considered. Possible mechanisms of ATP release will be tested in Aim 2. Demonstration of a role of specific channels in mechanical load-induced ATP release would be a major advance in understanding how chondrocytes respond to mechanical load. Specific Aim 3 is concerned with determining how extracellular signaling molecules, such as ATP, affect normal chondrocyte metabolism. In Specific Aim 4 we will determine how purinoceptors are regulated in chondrocytes. The results from the proposed studies will advance our understanding of the source of extracellular ATP in cartilage and provide a better understanding of how chondrocytes adapt to mechanical stimuli.